Source: UNIVERSITY OF CALIFORNIA, RIVERSIDE submitted to NRP
ENHANCING PLANT IMMUNITY BY MODULATING DEFENSE GENE EXPRESSION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0201309
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Oct 1, 2014
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
Botany and Plant Sciences
Non Technical Summary
An important goal of plant breeding and biotechnological research is the improvement of disease resistance of economically important crops. Plant diseases are the cause for dramatic losses in crop production. To defend themselves against microbial attack, plants have evolved a defense system that is inducible upon pathogen recognition. The overall goal of this project is to identify key regulatory mechanisms controlling plant immune responses and their manipulation to design new strategies to improve disease resistance in crops. Our understanding of the plant immune system has now progressed to a level that allows us to design gene-transfer-based approaches specifically tailored to enhance immunity of plants to defined pathogens. Due to the low consumer acceptance of gene-transfer-based approaches, however, many biotechnology companies are now also turning towards the design of novel agrochemicals to improve the performance of crop varieties. Given our expertise regarding the molecular genetics of the plant immune system, mechanisms of defense gene regulation and chemical genomics, my lab is in a unique position to make a major contribution to the design of new types of pesticides that fight plant diseases by enhancing the plant's inherent defense capabilities. Given the large amount of knowledge that has accumulated in recent years regarding transcriptional regulators of plant immune responses, we will also pursue gene-transfer-based approaches to manipulate the regulation of the defense transcriptome. As a main outcome we plan to provide a set of new chemical that induce defined immune responses in Arabidopsis and tomato as well as additional crop systems. These will serve as leads for the design of new pesticides. The majority of pesticides currently in use exhibit direct antibiotic activity which often leads to undesirable toxic environmental effects. Compounds that elicit innate plant immune responses offer an attractive alternative to application of toxic pesticides for disease control regimes and may allow for a more efficient and environmentally friendly protection of crops.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2012499104015%
2012499108015%
2012499200010%
2122499104020%
2122499108020%
2122499200020%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1080 - Genetics; 2000 - Chemistry;
Goals / Objectives
Several attempts have been made to increase disease resistance in crops by over-expressing single components of known defense pathways. Although gradually improved resistance could be achieved, so far the development of genetically modified crops based on this strategy has been a failure. Constitutive activation of defense responses in such GM crops severely compromised other plant functions leading to lower yields. A possible solution could be to tightly control ectopic expression of a defense component by highly specific promoters. Ideally such promoters would be strictly specific to pathogen infections. They should have no background activity or respond to other stresses, but should respond strongly to a wide range of virulent pathogens. Alternatively chemical inducers of defense gene expression could be used to achieve controlled and conditional expression of endogenous defense genes or transgenes. The design of either type of strategy requires detailed knowledge of key regulatory steps in the activation of the plant defense transcriptome. We identified an important regulatory mechanism of the Arabidopsis defense network, that is dependent on the WRKY70 transcription factor and which targets the promoter of the CaBP22 gene as well as other co-regulated defense genes. This node can be targeted by the synthetic elicitor DCA. Our results with transgenic -333_CaBP22::GUS tomato plants show that this DCA inducible mechanisms is conserved between Arabidopsis and tomato. One of our main goals for the second funding period of this project is to design transgene-based approaches to achieve heightened disease resistance by conditional activation of this WRKY70/CaBP22 module in Arabidopsis, tomato and additional crop systems, such as pepper and rice. Besides this we plan to identify other regulatory nodes of the plant defense network that can be manipulated to increase disease resistance in crops. A second major goal will be to utilize synthetic elicitors independently from transgenes and in combinations with transgenes to achieve enhanced disease resistance in crop systems and Arabidopsis. We propose the following two main objectives: (1)Design of transgene-based approaches to conditionally activate defense reactions in Arabidopsis, tomato and other crop systems (a using components of the WRKY70-dependent pathway of Arabidopsis (b)using components of other nodes of the Arabidopsis defense network. (2) Use of synthetic elicitors (or combinations thereof) to induce heightened disease resistance in (a) wild type accessions of Arabidopsis, tomato and other crop systems (b) transgenic lines of Arabidopsis or tomato containing elicitor-responsive transgenes.
Project Methods
1a: Transgenes will be cloned containing the Arabidopsis WRKY70 gene controlled by the LURP1 promoter, which does not exhibit any detectable levels of background activity and mediates a particularly strong relative increase of transcription in response to defense stimuli as well as the CaBP22 promoter, which we already showed to function in tomato. These constructs will be transformed into wild type plants of the Arabidopsis accession Col-0 and the tomato cultivar moneymaker. The resulting lines will be tested for reduced susceptibility to virulent pathogens. If results with the LURP1 and CaBP22 promoters are not satisfactorily, we will test include additional promoters in this study, such as the HARG1 promoter, which we identified by enhancer trapping. This promoter acts in a highly specific manner only responding in cells that are in direct contact with Peronospora hyphae. In addition, we will also test additional pathogen-responsive enhancer traps that we identified in Arabidopsis. Lines that exhibit GUS activity in a highly pathogen-specific manner, while showing no or low background activity, will be further examined. Their respective pathogen-responsive promoter regions will be isolated and used to drive WRKY70 expression in Arabidopsis and tomato. 1b: We are in the process of characterizing additional regulatory proteins of the Arabidopsis defense network that may be suitable targets for strategies to enhance disease resistance in crops. Transgenes will be constructed containing the promoters described above driving expression of the respective regulator in Arabidopsis and tomato. 2: Use of synthetic elicitors (or combinations thereof) to induce heightened disease resistance: We are currently performing screens for chemical elicitors of the pathogen-responsive the -333_CaBP22::GUS reporter gene in Arabidopsis. In addition we will initiate screens using other reporter systems, such as the HARG1 enhancer trap line. All potent inducers of these reporter genes identified by our screens with chemical structures distinct from known defense inducers will be further characterized. Using Arabidopsis and tomato we will test, if they (1) induce expression of endogenous defense-related genes, (2) induce disease resistance to virulent Peronospora and P. syringae, (3) determine the effects of known Arabidopsis or tomato defense mutations on their activity. Synthetic elicitors that, based on their analysis in Arabidopsis and tomato defense mutants, interfere with different parts of the defense network, will be tested for synergistic effect, when applied in combination. 2b: A possibly interesting variant of the strategies described above could be to combine the approaches outlined under points 1 and 2. Chimeric pathogen-inducible transgenes may respond with elevated expression to treatment with one of our synthetic elicitors. Thus the potency of this elicitor to induce enhanced disease resistance may be enhanced in Arabidopsis or tomato lines containing one of these transgenes. We will test this concept for a variety of transgene/elicitor combinations.

Progress 10/01/09 to 10/01/14

Outputs
Target Audience: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Using RSAP funds we provided training to the following individuals: - Erika Olson (undergraduate working in Dr. Eulgem's lab) - Eric Yang (undergraduate working in Dr. Eulgem's lab) - Johnathan Weiz (undergraduate working in Dr. Eulgem's lab) - Alex Park (undergraduate working in Dr. Eulgem's lab) - Natalie Williams (undergraduate working in Dr. Eulgem's lab from 2009 to 2012; from 2012 to 2014 graduate student with Dr. Eulgem - Mercedes Schroeder (junior specialist working with Dr. Eulgem from 2009 to 2011; from 2011 to 2014 graduate student with Dr. Eulgem) - Melinda Salus (graduate student of Dr. Eulgem) - Kishor Bhattarai (graduate student partially co-supervised by Drs. Eulgem and Kaloshian) - Ayesha Baig (graduate student of Dr. Eulgem) - Dr. Tokuji Tsuchiya (Assistant Specialist in Dr. Eulgem's lab) How have the results been disseminated to communities of interest? Results from this study have been presented in the following 23 invited talks: - "Dissecting plant defense signaling by chemical and molecular genetics" June 29th , 2010, Institute of Molecular Biology and Biotechnology/Heraklion University, Heraklion, Greece. - "Dissecting plant defense signaling by chemical and molecular genetics" July 7th , 2010, Institute of Agrobiotechnology, Thessaloniki, Greece - "Dissecting plant defense signaling by chemical and molecular genetics", December 12th, 2010, Fujian Agriculture and Forestry University; Fuzhou, Fujian Provience,PR China - "EDM2, a transcriptional regulator involved in distinct regulatory modules in Arabidopsis immunity & development " Dec. 14th, 2010. Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan - "EDM2, a transcriptional regulator involved in distinct regulatory modules in Arabidopsis immunity & development " Dec. 15th. 2010 Academia Sinica, Taipei, Taiwan - "EDM2, a transcriptional regulator involved in distinct regulatory modules in Arabidopsis immunity and development" seminar at Department of Cell & Systems Biology, University of Toronto Department of Cell & Systems Biology, University of Toronto, Toronto, Canada, 06/2011 - "Chemical Genomics of the Plant Immune System and Development of Novel Non-Biocidal Reduced Risk-Pesticides". Plant and Animal Genome XX Conference, San Diego, CA. Presented January 15, 2012 - "Chemical genomics of the plant immune system and development of novel non-biocidal reduced-risk pesticides", UCR Biochemistry department, regular seminar, February 28th, 2012., University of California at Riverside, CA, USA - "Chemical genomics of the plant immune system and development of novel non-biocidal reduced-risk pesticides", UCR Nematology department, regular seminar, May 12th, 2012, University of California at Riverside, CA, USA - "Synthetic elicitors as specific inducers of plant immune responses"' UCR IGERT Symposium June, 15th, 2012, University of California at Riverside, CA, USA - "Synthetic elicitirs as specific inducers of plant immune responses", September 15th, 2012, Fujian Agriculture and Forestry University; Fuzhou, Fujian Provience,PR China - "Synthetic elicitirs as specific inducers of plant immune responses", September 15th, 2012, Fujian Agriculture and Forestry University; Fuzhou, Fujian Provience,PR China - "EDM2, an Arabidopsis regulator of transposon activity involved in innate immunity and development" September 17th, 2012, Fujian Agriculture and Forestry University; Fuzhou, Fujian Provience,PR China - "EDM2, an Arabidopsis regulator of transposon activity involved in innate immunity and development" September 19th, 2012, Institute for Plant Ecology and Physiology, Shanghai, PR China - "EDM2, an epigenetic regulator of transposon activity controlling innate immunity & development in Arabidopsis", Regular Seminar at Institute of Biology, Dept. of Genetics Martin- Luther- University Halle Institute of Biology, Dept. of Genetics Martin- Luther- University Halle, Martin- Luther- University Halle, Germany, 02/2013 - "EDM2, an epigenetic regulator of transposon activity controlling innate immunity & development in Arabidopsis" Regular Seminar at Max-Planck Institute for Plant Breeding Research, Max-Planck Institute for Plant Breeding Research, Cologne, Germany 02/2013 - "EDM2, an epigenetic regulator of transposon activity controlling innate immunity & development in Arabidopsis", 26. Tagung Molekularbiologie der Pflanzen (Conference on Plant Molecular Biology), Deutsche Botansiche Gesellschaft (German Botanical society) Dabringhausen, Germany. 02/2013 - "EDM2, an epigenetic regulator of transposon activity controlling innate immunity & development in Arabidopsis", Regular Seminar at Center for Biotechnology, University of Bielefeld, Germany, Center for Biotechnology, University of Bielefeld, Germany, Bielefeld, Germany 03/2013 - "Regulation of the plant defense transcriptome", Seminar at College of Crop Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China, College of Crop Sciences, Fujian Agriculture and Forestry University, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China, 03/2013 - "A Transposon-associated Mechanism Controlling, Disease Resistance Gene Expression in Arabidopsis" at annual symposium of UCR Center for Plant Cell Biology, UC-Riverside, December 18, 2013 - "The Arabidopsis PHD-Finger Protein EDM2 Controls Plant Innate Immunity by Modulating Levels of the Epigenetic Transposon-Silencing Mark H3K9me2" AAAS, Pacific Division, 95th Annual Meeting University of California RIVERSIDE, CALIFORNIA, Small RNA-Mediated Gene Regulation Symposium, UC-Riverside, June 19, 2014 - "Transposon-associated mechanisms of NB-LRR immune receptor expression control" XVI International congress on Molecular Plant Microbe Interactions, July, 7, 2014, Rhodes, Greece - "Transposon-associated mechanisms of NB-LRR immune receptor expression control", special seminar at Freie Universität Berlin, Institut für Biologie & Angewandte Genetik, Berlin, Germany, July 24, 2014 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The provided HATCH funding allowed us to make substantial progress regarding the five areas outlined below, which are directly related to the goals of this project. These data resulted in 12 new publications, two patent applications and numerous seminar presentations and enabled us to recruit 2 NSF grants and a UCR Collaborative Seed Grant. Two additional NSF grant applications that are based on data from our HATCH project are currently pending. 1) Collaborative work we did with Professor isgouhi Kalosians lab (Department of nematology, UCR) showed that the transcription factors (TFs) WRKY70 and WRKY72 are structurally and functionally conserved between Arabidopsis and tomato. Both TFs mediate basal defense against several types of virulent pathogens/pests in either one of these species. For WRKY70 we could show that its role in mediating salicylic acid-dependent defense responses and suppressing jasmonic acid-dependent defense responses is preserved in both tested species. By high resolution time course transcript profiling using RNA-seq we also found WRKY70 type TF genes to be among the first genes to be up-regulated upon activation of salicylic acid signaling. In both species WRKY70-like genes exhibited similar induction kinetics. For WRKY72 we could show that it seems to operate in a salicylic acid- (and likely jasmonic acid-) independent manner in inducing immunity. Thus, both types TFs play distinct roles in defense signaling. The fact that they are structurally and functionally conserved between the distantly related eudicots A. thaliana and tomato suggests that they play similar roles across all eudicts. Therefore, combined overexpression or conditional expression of both TFs is likely to allow for the activation of a broad set of immune responses and to provide strong broad-spectrum pathogen resistance in eudicot crops. This strategy will be tested in the future. This work was published in or papers by Bhattarai et al., 2010 and Atamian et al., 2012. 2) Collaborative work we did with Prof. Shuilin He's lab (Fujian Agriculture and Forestry University, Fuzhou, China) on roles of WRKY transcription factors in crop disease protection showed that the TFs CaWRKY27, CaWRKY40 and CaWRKY58 regulate immune responses in pepper and that overexpression of these TFs class affects immunity in tobacco. While overexpression of CaWRKY27 and CaWRKY0 results in enhanced pathogen resistance, overexpression of CaWRKY58 suppresses immune responses in tobacco. This work resulted in our joint publications by Dang et al., 2013a, Dang et al., 2013b and Want et al, 2013. 3) We continued with the analysis of synthetic elicitor candidates originally identified in our high throughput screen for inducers of expression of a pathogen-responsive reporter gene in Arabidopsis (Knoth et al., 2009, Plant Physiol. 150: 333-347). We specifically focused on the characterization of members of the PTC and PMP classes of synthetic elicitors. We found that several PTC and PMP derivatives can induce immune responses in Arabidopsis as well as the crop species tomato and cowpea. In contrast to DCA, which interferes with a target operating causally downstream from SA, the PTC representative CMP442 interferes with a target that acts very early in defense signaling and controls the accumulation of SA We further tested the activity of one PMP derivative, CMP199, in various Arabidopsis defense mutants. This compound seems not to require any of the known defense regulators acting in the canonical salicylic acid response pathway. This is surprising, as CMP199 induces expression of known SA response marker genes. Manuscripts on PTCs and PMPs are in a final stage of preparation. In addition patent applications for both PTCs and PMPs as new crop protection compounds have been filed. A book chapter outlining our chemical screening strategy has recently been published (Knoth & Eulgem, 2014). 4) While defense inducing compounds generally tend to reduce plant growth and biomass when applied at high doses, we surprisingly found that low doses of some synthetic elicitors we identified stimulate enhanced growth of roots and aerial plant parts. At these low doses they still can induce defense reactions. This interesting additional effect on developmental processes makes our synthetic elicitors even more attractive candidates for strategies to improve disease resistance of crops. We will take advantage of this phenomenon to design novel chemistry-based strategies to accelerate crop plant development while at the same time boosting their immunity. We characterized this phenomenon at the transcriptome level and found that synthetic elicitor-mediated growth enhancement is linked to the stimulation of root specific gene expression. Both known defense signaling components and regulators of responses required for developmental processes controlled by the phytohormone auxin are needed for synthetic elicitor-triggered hormesis. A manuscript on this phenomenon is in an advanced stage of preparation. 5) We also uncovered in my lab a novel epigenetic mechanism in Arabidopsis that is critical for the regulation of at least two (but likely more) immune receptor genes. This mechanism is dependent on the chromatin-assoiated factor EDM2 and the epigenetic silencing mark H3K9me2. By modulating H3K9me2 levels, Arabidopsis plants can fine tune the use of alternative polyadenylation sites in in the immune receptor genes RPP4 and RPP7 as well as likely in a dditional defense related gene. We further found H3K9me2 levels to be altered in response to pathogen recognition and identified several mutants that are compromised in this response. HATCH funding allowed us to generate data for six research articles on this new mechanism and EDM2 (Tsuchiya & Eulgem, 2010a, 2010b, 2011, 2013a, 2013b and 2014).

Publications

  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Tsuchiya T, Eulgem T. 2010. The Arabidopsis defense component EDM2 affects the floral transition in an FLC-dependent manner. The Plant Journal. 62: 518-28
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Bhattarai KK, Atamian HS, Kaloshian I, Eulgem T. 2010. WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-for-gene resistance mediated by the tomato R gene Mi-1. The Plant Journal 63: 229-40
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Tsuchiya T, Eulgem T. 2010. Co-option of EDM2 to distinct regulatory modules in Arabidopsis thaliana development. BMC Plant Biology 10: 203
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Tsuchiya T, Eulgem T., 2011. EMSY-like genes are required for full RPP7-mediated race specific immunity and basal defense in Arabidopsis. Molecular Plant Microbe Interactions, 24: 15731581
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Atamian, H.S, Eulgem, T., Kaloshian, I., 2012. SlWRKY70 is required for Mi-1-mediated resistance to aphids and nematodes in tomato. Planta, 235: 299309
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Feng-Feng Dang, Yu-Na Wang, Lu Yu, Thomas Eulgem, Yan Lai, Zhi-Qin Liu, Xu Wang, Ai-Lian Qiu, Ting-Xiu Zhang, Jing Lin, Yan-Sheng Chen, De-Yi Guan, Han-Yang Cai, Shao-Liang Mou, Shui-Lin He. 2013. CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection. Plant, Cell & Environment, 36: 757-774
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Yuna Wang, Fengfeng Dang, Xu Wang, Thomas Eulgem, Yan Lai, Lu Yu, Zhiqin Liu, Jianju She, Youliang Shi, Jinhui Lin, Chengcong Chen, Deyi Guan, Ailian Qiu & Shuilin He. 2013. CaWRKY58 encoding a group I WRKY Transcription Factor of Capsicum annuum Negatively Regulates Resistance to Ralstonia solanacearum Infection. Molecular Plant Pathology, 14: 131-144
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Tokuji Tsuchiya & Thomas Eulgem. 2013. Mutations in EDM2 selectively affect silencing states of transposons and induce plant developmental plasticity. Scientific Reports, 3: 1701
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Tokuji Tsuchiya & Thomas Eulgem. 2013. An alternative polyadenylation mechanism coopted to the Arabidopsis RPP7 gene through intronic retrotransposon domestication. Proc Natl Acad Sci USA, 110:E3535E3543.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Sakhon, O., Victor, K., Choy, A., Tsuchiya , T., Eulgem, T., Pedra, J. 2013. NSD1 Mitigates Caspase-1 Activation by Listeriolysin O in Macrophages. PLoS ONE. Vol. 8: 9- 12
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Tokuji Tsuchiya & Thomas Eulgem. 2013.The PHD-finger module of the Arabidopsis thaliana defense regulator EDM2 can recognize triply modified histone H3 peptides. Plant Signal Behav. 9. pii: e29202.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Knoth, Colleen; Eulgem, Thomas, 2014 High-Throughput Screening of Small-Molecule Libraries for Inducers of Plant Defense Responses in PLANT CHEMICAL GENOMICS: METHODS AND PROTOCOLS Edited by: Hicks, GR; Robert, S; Book Series: Methods in Molecular Biology Volume: 1056 Pages: 45-49
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Dang, F., WANG , Y., She, J., Lai, Y., LIN, J., Eulgem, T., LIU, Z., YU, L., Lei, Y., GUAN, D., Lei, D., Li , X., Yuan, Q., HE, S. 2013. Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection. Physiol. Plant. doi: 10.1111/ppl.12093 29p


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We provided during the current evaluation period training to the following individuals: - Dr. Tokuji Tsuchiya (Assistant Specialist in Dr. Eulgem’s lab) - Mercedes Schroeder (graduate student of Dr. Eulgem) - Yasemin Bektas (graduate student of Dr. Eulgem) - Natalie Williams (graduate student of Dr. Eulgem) - Adilene Gomez (undergraduate working in Dr. Eulgem’s lab) -Jesus Banderas (undergraduate working in during summer 2013 in Dr. Eulgem’s lab) How have the results been disseminated to communities of interest? Results from our study have been disseminated to communities of interest via six peer-reviewed technical research articles and five seminar/conference presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In collaboration with Professor Shuilin He’s lab at the Fujian Agriculrtural and Forestry University (Fuzhou, China) we demonstrated that overexpression of the transcription factors CaWRKY27 and CaWRKY40 mediates constitutive resistance of tobacco against the bacterial pathogen Ralstonea solanacearum. We also uncovered in my lab an epigenetic mechanism in Arabidopsis that is critical for the regulation of expression of the immune receptor RPP7. This mechanism is dependent in the epigenetic regulator EDM2 as well as the epigenetic silencing mark H3K9me2. By modulating H3K9me2 levels in the first intron of RPP7 gene, Arabidopsis plants fine tune the use of an alterantive polyadenylation site and control levels of functional RPP7 coding transcripts. This mechanism can be exploited to artificially modify expression levels of plant defense genes. We found the synthetic elicitor HTC to suppress disease symptoms caused by the fungal pathogen Fusarium oxysporum in the legume cowpea. In a large greenhouse study using 100 plants per treatment group we found repeated application of 500 uM HTC to significantly reduce the intensity of disease symptoms by 10%. We also compared the biological activities of the synthetic elicitors DCA, HTC and CMP199 in a set of known Arabidopsis defense mutants and found them to differ in their mode of defense induction. Thus with DCA, HTC and CMP199 we have identified functionally distinct synthetic elicitors with the potential to protect crop species from diseases under lab and greenhouse conditions. We also found CMP199 to be the most potent candidate of those synthetic elicitors we so far characterized. CMP199 belongs to the class of Phenylimino-methyl-phenols (PMPs). By pre-screening of 17 additional PMP derivatives in Arabidopsis we then identified two compounds that are equally or more potent that CMP199 in suppressing spore production of the pathogenic ooycete Hyaloperonospora arabidopsidis.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: 1) Dang, F., WANG , Y., YU, L., Eulgem, T., Lai, Y., LIU, Z., WANG, X., QIU, A., ZHANG, T., LIN, J., CHEN, Y., GUAN, D., CAI, H., MOU, S., HE, S. 2013. CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection. Plant, Cell & Environment. Vol. 36: p.757-774 . (Refereed) 2) WANG , Y., Dang, F., WANG, X., Eulgem, T., Lai, Y., LIU, Z., She, J., Shi, Y., Chen , C., GUAN, D., QIU, A., HE, S. 2013. CaWRKY58 encoding a group I WRKY Transcription Factor of Capsicum annuum Negatively Regulates Resistance to Ralstonia solanacearum Infection. Molecular Plant Pathology. Vol. 14: p.131-144. (Refereed) 3) Tsuchiya , T., Eulgem, T. 2013. Mutations in EDM2 selectively affect silencing states of transposons and induce plant developmental plasticity. Scientific Reports. Vol. 3: 9p. (Refereed) 4) Tsuchiya , T., Eulgem, T. 2013. An alternative polyadenylation mechanism coopted to the Arabidopsis RPP7 gene through intronic retrotransposon domestication. Proc Natl Acad Sci USA. Vol. 110: p.E3535E3543. (Refereed) 5) Sakhon, O., Victor, K., Choy, A., Tsuchiya , T., Eulgem, T., Pedra, J. 2013. NSD1 Mitigates Caspase-1 Activation by Listeriolysin O in Macrophages. PLoS ONE. Vol. 8: 9 12p. (Refereed) 6) Dang, F., WANG , Y., She, J., Lai, Y., LIN, J., Eulgem, T., LIU, Z., YU, L., Lei, Y., GUAN, D., Lei, D., Li , X., Yuan, Q., HE, S. 2013. Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection. Physiol. Plant. doi: 10.1111/ppl.12093 29p. (Refereed)


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. Using RSAP funds we provided in 2012 training to the following individuals: - Dr. Tokuji Tsuchiya (Assistant Specialist in Dr. Eulgem's lab) - Mercedes Schroeder (graduate student of Dr. Eulgem) - Ayesha Baig (graduate student of Dr. Eulgem) - Yasemin Bektas (graduate student of Dr. Eulgem) - Natalie Williams (graduate student of Dr. Eulgem) - Erika Olson (undergraduate working in Dr. Eulgem's lab) Results from this study have been presented in the following invited talks: - Chemical Genomics of the Plant Immune System and Development of Novel Non-Biocidal Reduced Risk-Pesticides. Plant and Animal Genome XX Conference, San Diego, CA. Presented January 15, 2012 - Chemical genomics of the plant immune system and development of novel non-biocidal reduced-risk pesticides, UCR Biochemistry department, regular seminar, February 28th, 2012., University of California at Riverside, CA, USA - Chemical genomics of the plant immune system and development of novel non-biocidal reduced-risk pesticides, UCR Nematology department, regular seminar, May 12th, 2012, University of California at Riverside, CA, USA - Synthetic elicitors as specific inducers of plant immune responses UCR IGERT Symposium June, 15th, 2012, University of California at Riverside, CA, USA - "Synthetic elicitirs as specific inducers of plant immune responses", September 15th, 2012, Fujian Agriculture and Forestry University; Fuzhou, Fujian Provience,PR China - "Synthetic elicitirs as specific inducers of plant immune responses", September 15th, 2012, Fujian Agriculture and Forestry University; Fuzhou, Fujian Provience,PR China - "EDM2, an Arabidopsis regulator of transposon activity involved in innate immunity and development" September 17th, 2012, Fujian Agriculture and Forestry University; Fuzhou, Fujian Provience,PR China - "EDM2, an Arabidopsis regulator of transposon activity involved in innate immunity and development" September 19th, 2012, Institute for Plant Ecology and Physiology, Shanghai, PR China PARTICIPANTS: - Dr. Thomas Eulgem (PI): supervised work of assistant specialists, graduate students and undergraduates working on this project and performed bioinformatics analyses. - Dr. Isgouhi Kaloshian (collaborator; Professor of UCR Nematology department): We collaborated extensively with Dr. Kaloshian on the function of synthetic elicitors in tomato as well as WRKY transcription factors in tomato. - Dr. Philip Roberts (collaborator; Professor of UCR Nematology department): We collaborated extensively with Dr. Roberts on the function of synthetic elicitors in the legume system cow pea. - Dr. Shuilin He (collaborator, Professor Fujian Agriculture and Forestry University; Fuzhou, Fujian Provience,PR China): We collaborated extensively with Dr. He on the function of synthetic elicitors in the crop systems rice, pepper and tobacco. - Dr. Tokuji Tsuchiya (Assistant Specialist working with Dr. Eulgem); performed molecular genetics analyses of the epigenetic transcriptional regulator EDM2 in Arabidopsis. - Mercedes Schroeder (graduate student of Dr. Eulgem): Characterization of synthetic elicitors DCA, PTCs and PMPs in crop systems. - Ayesha Baig (graduate student of Dr. Eulgem); tested synthetic elicitors in tomato. Overexpression of the Arabidopsis LURP1 and LOR1 genes. Analysis of LURP1 and LOR1 function in disease resistance. - Yasemin Bektas (graduate student of Dr. Eulgem); tested synthetic elicitors in tomato. Characterized effects on PTCs on plant growth. - Natalie Williams (graduate student of Dr. Eulgem); performed reverse genetic experiments with Arabidopsis lines containing mutations in genes responsive to the synthetic elicitor DCA. - Erika Olson (undergraduate working in Dr. Eulgem's lab); assisted Natalie Williams in reverse genetics analyses TARGET AUDIENCES: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1) We continued with the analysis of synthetic elicitor candidates originally identified in our high throughput screen for inducers of expression of a pathogen-responsive reporter gene in Arabidopsis (Knoth et al., 2009, Plant Physiol. 150: 333-347). We specifically focused on the characterization of members of the PTC and PMP classes of synthetic elicitors. We found that several PTC and PMP derivatives can induce immune responses in Arabidopsis. A manuscript on PTCs is in a final stage or preparation. We further tested the activity of one PMP derivative, CMP199, in various Arabidopsis defense mutants. This compound seems not to require any of the known defense regulators acting in the canonical salicylic acid response pathway. This is surprising, as CMP199 induces expression of known SA response marker genes. We are continuing with the analysis of this interesting compound and its derivatives in Arabidopsis and several crop systems. 2) We also continued to analyze hormetic effects we observed with low doses of several synthetic elicitors. While these compounds inhibited plant growth when applied at high doses, we found very low doses of them to stimulate plant growth. We observed these effects in Arabidopsis and tomato. We further found that under conditions of stress such hormetic effects are stronger. For example, when limited amounts of sucrose were provided to the growth medium, we found the PTC compound HTC to increase the length of Arabidopsis roots by over 100%, while under standard sucrose conditions only 40 - 50% increases of root growth were observed. We established experimental conditions that will allow us to profile transcriptional changes associated with hormetic effects induced by the synthetic elicitors CMP199 and HTC. These experiments will be performed in the first half of 2013. 3) We have been progressing by reverse genetic analyses of a subset of genes responsive to the synthetic elicitor DCA. We have been specifically focusing on two members of this set, which encode a novel type of defense-associated protein kinase containing a malectin-like domain. We found these protein kinases to be required for basal defense to our model pathogen Hyaloperonospora arabidopsidis (Hpa). We have started to make several constructs that will allow co-silencing of members of this protein kinase family as well as determining their subcellular localization.

Publications

  • No publications reported this period


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. Using RSAP funds we provided in 2011 training to the following individuals: - Dr. Yu-Hung Wei (part time Assistant Specialist in Dr. Eulgem's lab) - Dr. Tokuji Tsuchiya (Assistant Specialist in Dr. Eulgem's lab) - Melinda Salus (graduate student of Dr. Eulgem) - Mercedes Schroeder (graduate student of Dr. Eulgem) - Ayesha Baig (graduate student of Dr. Eulgem) - Yasemin Bektas (graduate student of Dr. Eulgem) - Johnathan Weiz (undergraduate working in Dr. Eulgem's lab) - Natalie Williams (undergraduate working in Dr. Eulgem's lab) - Erika Olson (undergraduate working in Dr. Eulgem's lab) Results from this study have been presented in the following invited talk: - EDM2, a transcriptional regulator involved in distinct regulatory modules in Arabidopsis immunity and development seminar at Department of Cell & Systems Biology, University of Toronto, Department of Cell & Systems Biology, , Canada 06/2011 PARTICIPANTS: - Dr. Thomas Eulgem (PI): supervised work of assistant specialists, graduate students and undergraduates working on this project and performed bioinformatics analyses. - Dr. Yu-Hung Wei (part time Assistant Specialist working with Dr. Eulgem); performed molecular genetics analyses of genes induced by synthetic elicitors in Arabidopsis and tomato. Performed functional characterization of the promoter of the disease resistance gene RPP7. - Dr. Tokuji Tsuchiya (Assistant Specialist working with Dr. Eulgem); performed molecular genetics analyses of the epigenetic transcriptional regulator EDM2 in Arabidopsis. - Melinda Salus (graduate student of Dr. Eulgem): performed chemical elicitor screens, participated in the characterization of DCA and characterized PTCs as well as other new synthetic elicitors. Designed and performed activation tagging screen for Arabidopsis genes required for elicitor activity. Tested T-DNA insertion lines of Arabidopsis ACID genes. Characterized effects of PTCs and oterh synthetic elicitors on Arabidopsis and tomato growth. - Mercedes Schroeder (graduate student of Dr. Eulgem): Characterization of synthetic elicitors DCA, PTCs and PMPs in crop systems. - Ayesha Baig (graduate student of Dr. Eulgem); tested synthetic elicitors in tomato. Overexpression of the Arabidopsis LURP1 and LOR1 genes. Analysis of LURP1 and LOR1 function in disease resistance. - Yasemin Bektas (graduate student of Dr. Eulgem); tested synthetic elicitors in tomato. Characterized effects on PTCs on plant growth. TARGET AUDIENCES: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. PROJECT MODIFICATIONS: We propose to shift the emphasis of this project from transgene-based- (objective I) to chemistry-based (objective II) approaches and prioritize the use of crop systems (tomato, cowpea) over Arabidopsis. The reasons for this are two-fold: 1) Due to the low consumer acceptance of transgene-based approaches for crop improvement many biotech companies are now turning towards the design of novel agrochemicals to improve crop performance. 2) Our tests using defense-inducing chemicals (synthetic elicitors) have been highly promising. Several of our candidates mediated clearly enhanced disease resistance in the crop systems tomato and cowpea. We also surprisingly discovered that low doses of the synthetic elicitor HTC enhanced plant growth leading to significantly longer roots and higher shoot fresh weight of tomato and Arabidopsis. Several biotech companies expressed interest in our synthetic elicitors. However, more proof-of-concept data for their applicability in crop protection are needed to recruit a private sector partner for a collaborative project. The proposed shift in our priorities is likely to provide these data. We still continue to pursue experiments addressing the utility of gene-transfer based manipulation of defense gene expression, as outlined under objective 1 in our original proposal. This goal is partly being followed by our collaboration with Prof. Shuilin He's lab at the Fujian University in China. In this collaborative project we could demonstrate that overexpression of two WRKY transcription factors enhances resistance of tobacco to the bacterial pathogen Ralstonia solanacearum. Manuscripts reporting on these observations have been submitted. In my lab we are also analyzing the effects of overexpressing the Arabidopsis LURP1 and LOR1 genes on disease resistance, which have been described in our previous publication by Knoth & Eulgem (2008; Plant J. 55: 53). However, we propose to more strongly pursue the characterization of synthetic elicitors as leads for the development of novel environmentally friendly pesticides that protect crops from diseases by inducing their natural immune system instead of being toxic for pathogens and harmful for other non-target organisms. In addition, the potential of HTC (and possibly additional synthetic elicitors) to enhance plant growth offers attractive perspectives for their use as agrochemicals. Therefore, we will expand objective II of our original proposal and perform extensive tests of synthetic elicitor candidates in the crop systems tomato and cowpea. Susceptibility of both species to agriculturally relevant pathogens will be tested after application of various synthetic elicitors. We will also examine effects of HTC and other synthetic elicitors on growth and vigor in cowpea and other plant systems, including the major staple cereal barley and the legume model Medicago truncatula. The reason for including the latter two species is that they offer excellent resources for genome-wide association mapping, which we consider to employ for the identification of plant genes required for the growth enhancing effects of HTC in future funding periods of this project.

Impacts
1) We continued with the analysis of synthetic elicitor candidates originally identified in our high throughput screen for inducers of expression of a pathogen-responsive reporter gene in Arabidopsis (Knoth et al., 2009, Plant Physiol. 150: 333-347). Recent experiments we performed established two distinct structural classes of small organic molecules as novel types of synthetic elicitors. Representatives of these two classes, provisionally termed PTCs and PMPs, efficiently activated immune responses in Arabidopsis against the virulent isolate Noco2 of the pathogenic oomycete Hyaloperonospora arabidopsidis (Hpa). A PTC representative also protected the legume cowpea from infection with the fungal pathogen Fusarium oxysporum. Based on differences in their biological activity in Arabidopsis defense mutants, we further found PTC and PMPs to differ in their mode-of-action from our previously described synthetic elicitor DCA (Knoth et al., 2009, Plant Physiol. 150: 333-347). A manuscript on PTCs is in a final stage or preparation and will be submitted to Plant Physiology in May 2012. 2) Previously we reported the identification of a set of 137 Arabidopsis genes that are co-induced by the synthetic elicitors DCA and INA (Knoth et al., 2009, Plant Physiol. 150: 333). We have been systematically testing these ACID (associated with chemically-induced defense) genes for their contribution to natural plant immune responses using Arabidopsis T-DNA mutants. So far we found 10 of 16 tested ACID genes to be required for basal defense against HpaNoco2. For each of these 10 genes two independent T-DNA mutants exhibited enhanced susceptibility to the virulent Hpa isolate Noco2. These results support that gene expression changes triggered by synthetic elicitors constitute authentic plant immune responses. Many genes that are co-induced by DCA and INA are indeed required for full plant immunity. Of the 10 ACID genes we found to be required for resistance to HpaNoco2, eight have not been described as components of the plant immune system yet. We are constructing double mutants combining mutations in some of these 10 ACID genes to uncover their genetic relationship to each other. Double homozygotes for those double mutants are currently being selected and will soon be used in HpaNoco2 defense assays. 3) As described in our previous publication by Knoth et al. (MPMI, 20:12-128) the WRKY70 transcription factor is required for the defense-associated expression of the Arabidopsis LURP gene cluster. In collaboration with Dr Kaloshian's lab we have confirmed that tomato orthologs of WRKY70 play a similar role in defense-responses of tomato. Using virus-induced gene silencing (VIGS) we found DCA-mediated induction of a LURP promoter GUS fusion to require each one of two WRKY70 orthologs of tomato. As reported in our recent paper by Atamian et al. (Planta, 235: 299-309), at least one WRKY70 ortholog is also needed for successful defense induction in tomato against aphids and nematodes.

Publications

  • - Tsuchiya T, Eulgem T., 2011. EMSY-like genes are required for full RPP7-mediated race specific immunity and basal defense in Arabidopsis. Molecular Plant Microbe Interactions, 24:1573.
  • - Atamian, H.S, Eulgem, T., Kaloshian, I., 2012; SlWRKY70 is required for Mi-1-mediated resistance to aphids and nematodes in tomato. Planta, 235:299; Published online: 7 September 2011


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. Using RSAP funds we provided in 2010 training to the following individuals: - Melinda Salus (graduate student of Dr. Eulgem) - Mercedes Schroeder (graduate student of Dr. Eulgem) - Ayesha Baig (graduate student of Dr. Eulgem) - Johnathan Weiz (undergraduate working in Dr. Eulgem's lab) - Natalie Williams (undergraduate working in Dr. Eulgem's lab) - Eric Yang (undergraduate working in Dr. Eulgem's lab) Results from this study have been presented in the following invited talks: - "Dissecting plant defense signaling by chemical and molecular genetics" June 29th , 2010, Institute of Molecular Biology and Biotechnology/Heraklion University, Heraklion, Greece. - "Dissecting plant defense signaling by chemical and molecular genetics" July 7th , 2010, Institute of Agrobiotechnology, Thessaloniki, Greece - "Dissecting plant defense signaling by chemical and molecular genetics", December 12th, 2010, Fujian Agriculture and Forestry University; Fuzhou, Fujian Provience,PR China - "EDM2, a transcriptional regulator involved in distinct regulatory modules in Arabidopsis immunity & development " Dec. 14th, 2010. Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan - "EDM2, a transcriptional regulator involved in distinct regulatory modules in Arabidopsis immunity & development " Dec. 15th. Academia Sinica, Taipei, Taiwan PARTICIPANTS: - Dr. Thomas Eulgem (PI): supervised work of graduate students on this project and performed bioinformatics analyses. - Melinda Salus (graduate student of Dr. Eulgem): performed chemical elicitor screens, participated in the characterization of DCA and characterized CMP442 as well as other new synthetic elicitors. Designed and performed activation tagging screen for Arabidopsis genes required for elicitor activity. Tested T-DNA insertion lines of elicitor inducible Arabidopsis genes. - Mercedes Schroeder (graduate student of Dr. Eulgem): performed screens of Arabidopsis enhancer trap lines and identified lines that are suitable for additional chemical screens. Helped with characterization of synthetic elicitors. - Ayesha Baig (graduate student of Dr. Eulgem); tested synthetic elicitors in tomato. - Dr. Isgouhi Kaloshian (collaborator; Professor of UCR Nematology department): We collaborated extensively with Dr. Kaloshian on the function ofsynthetic elicitors as well as WRKY transcription factors in tomato. TARGET AUDIENCES: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1) To identify Arabidopsis genes required for the biological activity of the synthetic defense elicitors we discovered, we have designed and performed an activation tagging-based high throughput screen in Arabidopsis. This approach is based on the random insertion in the Arabidopsis genome of T-DNA vectors with multiple transcriptional enhancers. These "activation tags" mediate over-expression of genes nearby their insertion sites. For this approach, we are taking advantage of the fact that Arabidopsis seedlings grown on medium containing high doses of synthetic elicitors exhibit substantially retarded root development. Using the Arabidopsis activation tagging population created by Weigel and co-workers (Weigel et al., 2000; Plant Physiol., 122: 1003) we screened for plant lines exhibiting enhanced reduction of root development on a moderate concentration of the synthetic elicitor CMP442. We identified seven independent lines that showed such increased responsiveness to CMP442. These lines are likely to contain activation tags near genes required for the uptake, stability, metabolization or perception of CMP442. We will identify the activation tag insertion sites for each of these seven lines and test candidate genes in their vicinity for enhanced expression. Positive candidate genes will be tested for their roles in elicitor perception and defense using Arabidopsis T-DNA mutants, virus induce gene silencing in tomato as well as stable over-expression of these genes in both Arabidopsis and tomato. Besides enhancing our understanding on the mode-of-action of synthetic elicitors, these experiments will provide interesting new genes with potential key roles in plant immunity. 2) While defense inducing compounds generally tend to reduce plant growth and biomass when applied at high doses, we surprisingly found that low doses of some synthetic elicitors we identified stimulate enhanced growth of roots and aerial plant parts. At these low doses they still can induce defense reactions. This interesting additional effect on developmental processes makes our synthetic elicitors even more attractive candidates for strategies to improve disease resistance of crops. We will take advantage of this phenomenon to design novel chemistry-based strategies to accelerate crop plant development while at the same time boosting their immunity. We will systematically test in tomato to what degree these elicitors influence root development, seedling vigor and other growth-related parameters. We will also quantify how they protect tomato from pathogens or pest. For a subset of particularly promising candidates we will establish an application regime (dose, timing and type of administration) that has beneficial effects on both plant growth and immunity in tomato. 3) Previously we reported the identification of a set of139 Arabidopsis genes that are induced by various synthetic elicitors (Knoth et al., Plant Physiol. 150: 333). We have been systematically testing these genes for their contribution to natural plant immune responses using Arabidopsis T-DNA mutants. So far this allowed us to identify two protein kinases as novel components of the plant immune system.

Publications

  • - Tsuchiya T, Eulgem T. 2010. The Arabidopsis defense component EDM2 affects the floral transition in an FLC-dependent manner. The Plant Journal. 62: 518-28. (peer-reviewed)
  • - Bhattarai KK, Atamian HS, Kaloshian I, Eulgem T., 2010. WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-for-gene resistance mediated by the tomato R gene Mi-1. The Plant Journal 63: 229-40. (peer-reviewed)
  • - Tsuchiya T, Eulgem T., 2010. Co-option of EDM2 to distinct regulatory modules in Arabidopsis thaliana development. BMC Plant Biology.;10(1):203. (peer-reviewed)


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Outputs: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. Using RSAP funds we provided in 2009 training to the following individuals: - Melinda Salus (graduate student of Dr. Eulgem) - Kishor Bhattarai (graduate student partially co-supervised by Drs. Eulgem and Kaloshian) - Mercedes Schroeder (junior specialist working with Dr. Eulgem) - Ayesha Baig (graduate student of Dr. Eulgem) - Johnathan Weiz (undergraduate working in Dr. Eulgem's lab) - Alex Park (undergraduate working in Dr. Eulgem's lab) Results from this study have been presented in the following invited talk: - "Dissecting plant defense signaling by chemical and molecular genetics" XIV International Congress on Molecular Plant-Microbe Interactions IS-MPMI Quebec City, Canada 07/2009 PARTICIPANTS: - Dr. Thomas Eulgem (PI): supervised work of graduate students on this project and performed bioinformatics analyses. - Melinda Salus (graduate student of Dr. Eulgem): performed chemical elicitor screens, participated in the characterization of DCA and characterized CMP_442 as well as other new synthetic elicitors - Mercedes Schroeder (graduate student of Dr. Eulgem): performed screens of Arabidopsis enhancer trap lines and identified lines that are suitable for additional chemical screens. Helped with characterization of DCA. - Dr. Isgouhi Kaloshian (collaborator; Associate Professor of UCR Nematology department): We collaborated extensively with Dr. Kaloshian on the function of the LURP2 promoter in tomato. - Dr. Kishor Bhattarai (graduate student of Dr. Kaloshian, co-supervised by Drs. Kaloshiana and Eulgem on his work on WRKY72 in tomato) TARGET AUDIENCES: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1) The recently completed tomato genome sequence revealed that WRKY70, a single copy gene in Arabidopsis, is triplicated in tomato. Virus-induced gene silencing (VIGS) experiments we performed in collaboration with Dr. Isgouhi Kaloshian's lab (UCR Dept. of Nematology) suggested that at least one of these three tomato WRKY70 paralogs acts as a repressor of defense, while at least one other paralog positively contributes to plant immunity. These findings slightly changed our plans, as it may be suitable to either specifically overexpress a defense-activating tomato WRKY70 paralog, or to specifically silence a defense-suppressing tomato WRKY70 paralog in order to achieve enhanced pathogen resistance. Originally we had planned to use overexpression of the Arabidopsis WRKY70 ortholog for this purpose. A manuscript describing our findings regarding SlWRKY70a is in an advanced state of preparation and will be submitted to a peer reviewed journal by the mid of March 2010. 2) In collaboration with Dr. Kaloshian's lab we found by VIGS two tomato orthologs of Arabidopsis WRKY72 to positively contribute to immunity. Analyses of Arabidopsis lines with mutations in AtWRKY72 showed that the defense function of WRKY72-type transcription factors is conserved between Arabidopsis and tomato. Microarray experiments we performed revealed that this transcription factor largely controls genes that are non-responsive to the defense hormone salicylic acid (SA). Thus, in contrast to WRKY70, which we found to act in a SA-dependent defense pathway in both Arabidopsis and tomato, WRKY72 appears to act in a parallel, SA-independent pathway. This observation is potentially very important, as it suggest that the combined overexpression of WRKY70- and WRKY72-type transcription factors may allow for the activation of a very broad set of plant immune responses. We are currently constructing Arabidopsis lines overexpressing the Arabidopsis orthologs of each of these two transcription factors. We will also cross the resulting lines to test for effects resulting from combined overexpression of these two transcription factors. A manuscript reporting on our results with WRKY72-type transcription factors has been provisionally accepted by the Plant Journal. 3) We have characterized several new synthetic elicitors in Arabidopsis wild type plants and defense mutants. One of them, provisionally termed compound (CMP)442, highly efficiently mediates immunity to Hyaloperonospora parasitica and the bacterial pathogen Pseudomonas syringae. In addition CMP442 has properties that differ from those of DCA, a synthetic elicitor we previously identified and characterized. In contrast to DCA, which interferes with (a) target protein(s) operating causally downstream from SA, CMP442 hits (a) target(s) that acts very early in defense signaling and control(s) the accumulation of SA. We are currently testing derivatives of CMP442 for their efficiency and will soon test their performance in tomato. A manuscript reporting on our results with the synthetic elicitor DCA has been published in "Plant Physiology" (Knoth et al., 2009).

Publications

  • - Evrard, A., Ndatimana, T., Eulgem, T. (2009) FORCA, a promoter element that responds to crosstalk between defense and light-signaling. BMC Plant Biology. Vol. 9: doi:10.1186/1471-2229-9-2
  • - Knoth, C., Salus, M.S., Girke, T., Eulgem, T. (2009) The Synthetic Elicitor 3,5-Dichloroanthranilic Acid Induces NPR1-Dependent and NPR1-IndependentMechanisms of Disease Resistance in Arabidopsis. Plant Physiology. 150: 333-347


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. In addition, we provided in 2008 training to the following individuals: - Colleen Knoth (graduate student of Dr. Eulgem) - Melinda Salus (graduate student of Dr. Eulgem) - Mercedes Schroeder (graduate student of Dr. Eulgem) - Ayesha Baig (graduate student of Dr. Eulgem) - Arif Mosavi (undergraduate working in Dr. Eulgem's lab) - Wilhelm Ong (undergraduate working in Dr. Eulgem's lab) - Prof. He Shuilin (visiting Professor). Results from this study have been presented on the following posters/ abstracts: - "Dissection of LURP-dependent defense mechanisms by molecular and chemical genetics" by Knoth, C., Salus, M.S., Girke, T., Eulgem, T. 2008 Keystone symposium on Plant Innate Immunity; Keystone Resort, CO. Presented on 02/2008. - "A suite of novel defense elicitors as tools for systems-level analyses of the plant defense network" by Salus, M.S., Knoth, C., Schroeder, M., Eulgem. ASPB (American society of Plant Biologists) Annual Meeting, June 26 - July 1, 2008, Merida, Mexico. Conference/Meeting Date: 06/26/2008. - "Dissecting Arabidopsis immune responses using molecular and chemical genomics" by Knoth, C., Salus, M.S., Girke, T., Eulgem, T.. ASPB (American society of Plant Biologists) Annual Meeting, June 26 - July 1, 2008, Merida, Mexico. ASPB (American society of Plant Biologists) annual meeting, June 26 - July 1, 2008, Merida, Mexico. Conference/Meeting Date: 06/28/2008. Results from this study have been presented in the following invited talk: - "Dissecting plant defense signaling by chemical and molecular genetics" 19th International Conference on Arabidopsis Research. Hyatt Regency: Montreal, Canada. Invited plenary talk presented by Thomas Eulgem on 07/2008. PARTICIPANTS: - Dr. Thomas Eulgem (PI): supervised work of graduate students on this project and performed bioinformatics analyses. - Colleen Knoth (graduate student of Dr. Eulgem): examined the roles of the LURP1 gene in disease resistance and characterized its promoter. Characterized DCA - Melinda Salus (graduate student of Dr. Eulgem): performed chemical elicitor screens and participated in the characterization of DCA - Mercedes Schroeder (graduate student of Dr. Eulgem): performed screens of Arabidopsis enhancer trap lines and identified lines that are suitable for additional chemical screens. Helped with characterization of CMP67. - Dr. Isgouhi Kaloshian (collaborator; Associate Professor of UCR Nematology department): We collaborated extensively with Dr. Kaloshian on the function of the LURP2 promoter in tomato. - Professor Shuilin He (collaborator; visiting Professor from Fujian Agriculture and Forestry University, Fuzhou,350002, Fujian Provience,PR China): worked with Ms. Schroeder on the characterization of enhancer trap lines. TARGET AUDIENCES: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The overall goal of this project is to identify regulatory mechanisms controlling the plant defense transcriptome as well as their use to design new strategies to improve disease resistance in crops. Our studies are based on microarray analyses of transcriptional responses triggered in Arabidopsis thaliana (Arabidopsis) by the pathogenic oomycete Hyaloperonospora parasitica (Peronospora). We also explored the use of enhancer-trapping for the discovery of Peronospora-responsive Arabidopsis genes. Throughout the project we aimed for the identification of the following four types of outcomes/impacts that may allow to engineer enhanced disease resistance of plants: (1) plant genes that mediate disease resistance; (2) plant promoters and promoter elements that respond to pathogen recognition; (3) plant transcription factors that interact with these promoters/elements and mediate defense-associated gene expression; (4) chemical elicitors of plant immune responses. In collaboration with Dr. Isgouhi Kaloshian (UCR department of Nematology) results from our studies in the model plant Arabidopsis are being translated to tomato, a crop system important for the local agriculture. In 2008 we published one peer reviewed research paper (Knoth & Eulgem, 2008). In addition, data generated by this project enabled us to recruit a new USDA grant (#003703-002). During 2008, we made progress regarding the following points: (I) We identified the Arabidopsis gene LURP1 as an important new component of the plant immune system. T-DNA insertions in this gene substantially reduced resistance of Arabidopsis to Peronospora. LURP1 transcript levels are strongly up-regulated after recognition of the pathogenic oomycetes Peronospora and Phytophthora infestans. We identified a W box-type element of the LURP1 promoter, WLURP1, to be important for this response. W-LURP1 interacts with nuclear protein factors. These interactions are partially dependent on the WRKY70 transcription factor. These results are described in our paper by Knoth and Eulgem (2008). (II) We continued with the characterization of 2,6-dichloroanthranilic acid (DCA, formerly designated as CMP67), which is related to the defense hormone salicylic acid (SA) and its analog INA. In contrast to SA and INA, which are fully dependent on NPR1, DCA is only partially dependent on NPR1. Thus, DCA appears to be a valuable novel tool, to trigger a new type of defense mechanisms, which are independent from the well characterized defense regulator NPR1. Microarray analyses revealed a cluster of 142 DCA- and INA-responsive genes, which constitute a core gene set associated with chemically induced disease resistance . (III) We made use of enhancer trapping to identify suitable pathogen-responsive promoters for chemical screens. We screened 11,000 Arabidopsis enhancer trap lines for Peronospora-induced enhancer trap activity. We identified several interesting lines that show reporter gene activity in plant tissue surrounding Peronospora hyphae, which is not inducible by DCA or salicylic acid. Thus, we have a set of new reporter lines in place to screen for additional synthetic elicitors that differ from DCA.

Publications

  • - Knoth, C. & Eulgem T. (2008) The oomycete response gene LURP1 is required for defense against Hyaloperonospora parasitica in Arabidopsis thaliana. The Plant Journal, 55: 53-64


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Results from this study have been presented on the following posters/abstracts on scientific conferences: - Knoth, C., Melinda, S., Girke, T., Eulgem, T. 2007; Mechanisms controlling coordinated transcriptional reprogramming of defense genes in Arabidopsis. Plant Biology Symposium 2007, January 18 - 20, 2007, Riverside CA. - Knoth, C., Melinda, S., Girke, T., Eulgem, T. 2007; Dissecting mechanisms controlling transcriptional reprogramming during Arabidopsis immune responses by molecular and chemical genomics. 2007 IGERT project meeting, May 13-15, 2007, Arlington, VA. - Knoth, C., Melinda, S., Girke, T., Eulgem, T. 2007; Dissecting Arabidopsis immune responses using molecular and chemical genomics. APS/SON joint meeting, July 28-August 1, 2007, San Diego, CA. Results from this study have been presented by Thomas Eulgem in the following invited talks: - San Diego Center for molecular Agriculture, Plant Biology Day, June 15, 2007 "Transcriptional re-programming during plant immune responses". - ASPB (American Society of Plant Biologists) annual meeting, July 7 - July 11, 2007, Chicago, IL, USA "EDM2, a novel transcriptional regulator mediating disease resistance in Arabidopsis thaliana". I served also as chair of the minisymposium on plant defense at this meeting. - "Alumni Day" Genetics Institute- University of Cologne, Germany, September, 13-14th, 2007, Cologne, Germany, "Transcriptional re-programming during plant immune responses". PARTICIPANTS: - Dr. Thomas Eulgem (PI): supervised work of graduate students on this project and performed bioinformatics analyses. - Colleen Knoth (graduate student of Dr. Eulgem): examined the roles of LURP genes in disease resistance and characterized two LURP promoters. Identified and characterized the LURP-A promoter element. Performed chemical screns for LURP inducers that lead to the identification CMP67. - Melinda Salus (graduate student of Dr. Eulgem): assisted Ms. Knoth with the chemical screens. - Mercedes Schroeder (graduate student of Dr. Eulgem): performed screens of Arabidopsis enhancer trap lines and identified lines that are suitable for additional chemical screens. Helped with characterization of CMP67. - Dr. Alexandre Evrard (postdoctoral scientis in Dr. Eulgem's laboratory): performed all experiments on the FORC-A promoter element. - Dr. Isgouhi Kaloshian (collaborator; Associate Professor of UCR Nematology department): We collaborated extensively with Dr. Kaloshian on the function of the LURP2 promoter in tomato. TARGET AUDIENCES: This project targets the plant pathology and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant pathology/plant immune biology. PROJECT MODIFICATIONS: One minor change relative to our original proposal is that we expanded the project by performing screens for chemicals inducing the new defense regulatory mechanisms that we have discovered. This new objective serves the main goal of our project by providing starting points for the development of new (pesticide-based) strategies to improve disease resistance of crops.

Impacts
The goal of this project is to identify regulatory mechanisms controlling the plant defense transcriptome as well as their use to design new strategies to improve disease resistance in crops. Our studies are based on microarray analyses of transcriptional responses triggered in Arabidopsis thaliana (Arabidopsis) by Hyaloperonospora parasitica (Peronospora). We focused on promoter motifs potentially involved in the regulation of two groups of co-expressed defense genes called FORCs (fungal and oomycete pathogen response cluster) and LURPs (late up-regulated in response to Peronospora recognition). As described in our previous progress reports, these motifs interacted in a Peronospora-dependent manner with nuclear Arabidopsis proteins in EMSAs. We further proved defense-related roles of two of these motifs, designated as FORC-A and LURP-A, using GUS-reporter gene fusions in stably transformed Arabidopsis plants or transiently transformed tobacco leaves. Our results indicate that FORC-A is a light responsive enhancer element, that suppresses gene expression in response to defense-related treatments. Our data established FORC-A as a novel element of crosstalk between light and defense signaling. We propose that FORC-A contributes to the early and transient Peronospora-induced up-regulation of FORC genes by mediating their down-regulation at later time points. In contrast to FORC-A, LURP-A proved in our reporter gene assays to be a positive pathogen-response element. In collaboration with Dr. Isgouhi Kaloshian (Department of Nematology, UCR) we further showed that the LURP-A containing LURP2 promoter fused to GUS (LURP2::GUS) responded to defense-stimuli in stably transformed tomato and Arabidopsis plants. Hence the LURP2 promoter is ubiquitously functional in multiple dicot species, including the agriculturally important tomato. To identify novel molecular tools to further examine regulatory mechanisms controlling LURP-dependent defense responses, we screened 44,000 diverse organic compounds of libraries generated by combinatorial chemistry for substances inducing LURP2::GUS expression in the absence of Peronospora or other defense-related stimuli. One novel compound was identified that acts as a potent activator of LURP2 expression and disease resistance to Peronospora and Pseudomonas syringae. This novel synthetic elicitor, CMP67, differs in its kinetic properties from known defense inducers, as it acts much faster and more transiently allowing real-time control over defense processes. We are currently characterizing the activity of CMP67 and other promising new elicitors identified by our screens in Arabidopsis and tomato. Our goal is to develop a set of novel synthetic elicitors that trigger disease resistance across plant species barriers and to use them as specific triggers for the fine dissection of the defense network as well as "leads" for the design of novel pesticides that protect plants by stimulating their inherent defense mechanisms.

Publications

  • - Knoth, C. Ringler, J. Dangl., J.L., Eulgem, T. (2007) Arabidopsis WRKY70 is required for full RPP4-mediated disease resistance and basal defense against Hyaloperonospora parasitica; Molecular Plant Microbe Interactions 20: 120-128.
  • - Eulgem, T., Tsuchiya, T., Wang, X.-J., Beasley, B., Cuzick, A., Toer, M., Zhu, T, McDowell, J.M., Holub,E. & Dangl, J.L; (2007) EDM2 is a novel component of RPP7-dependent disease resistance in Arabidopsis that affects RPP7 transcript levels; The Plant Journal 49: 829-839.
  • - Eulgem, T. & Somssich, I. E. (2007) Networks of WRKY Transcription Factors in Defense Signaling Current Opinion in Plant Biology, 10:366.


Progress 01/01/06 to 12/31/06

Outputs
The goal of this project is to identify novel cis-elements and transcription factors controlling the plant defense transcriptome as well as their use to design new strategies to improve disease resistance in crops. Our studies are based on microarray analyses of transcriptional responses triggered in Arabidopsis thaliana (Arabidopsis) by Hyaloperonospora parasitica (Peronospora). We focused mainly on conserved promoter motifs potentially involved in the regulation of two groups of co-expressed genes called EURPs (early up-regulated in response to Peronospora recognition) and LURPs (late up-regulated in response to Peronospora recognition). As described in our previous progress report some of these motifs (M1, M2, M3, M5, M6) interacted in a Peronospora-dependent manner with nuclear Arabidopsis proteins in EMSAs. To prove roles of these motifs in defense gene regulation, we constructed a set of binary reporter constructs consisting of a timer of each motif inserted upstream from the -46 CaMV35S minimal promoter and the GUS coding sequence. We created transgenic Arabidopsis lines harboring these constructs and established an Agrobacterium-mediated transient expression protocol to test them in tobacco. Using these tools, we are currently examining their functions in tobacco and Arabidopsis. Our preliminary results indicate that M3 is a negative element suppressing gene expression in response to pathogen infection and that M6 is a positive element, activating gene expression in response to defense-related stimuli. By mass-spectrometry with proteins purified by DNA-affinity chromatography, we identified putative M3-binding proteins. We are currently examining their interactions with M3. M3 may be useful in limiting the activity of pathogen responsive trans-genes mediating disease resistance, which may cause extensive damage of plant tissues. This possibility as well as potential roles of the identified M3 binding proteins in EURP regulation and disease resistance as well as the functions of additional conserved motifs will be addressed in experiments we have planned for 2007. Furthermore we have identified the Arabidopsis transcription factor WRKY70 as a key regulator of LURP gene expression. WRKY70 is tightly co-expressed with LURP genes. Two independent T-DNA insertions in the Arabidopsis WRKY70 gene resulted in reduced transctipt levels of LURP genes and reduced resistance to different Peronospora isolates. These results are described in a paper we recently published (Knoth et al., 2007, MPMI, 20: 120-128). In collaboration with Dr. Isgouhi Kaloshian (Department of Nematology, UCR) we have generated transgenic tomato lines that harbor a WRKY70-controlled LURP promoter fused to GUS. We will soon test if this promoter responds in tomato to a variety of pathogens, pests and other defense-related treatments.

Impacts
We are examining molecular mechanism of defense gene regulation. Findings from this study will be used to design new strategies to improve disease resistance in crops. Besides leading to increased crop production our strategy is also likely to lead to a reduction of pesticide use. The latter effect may substantially impact the quality of the natural environment and conservation of natural resources.

Publications

  • - Eulgem, T.; (2006) Dissecting the WRKY Web of Plant Defense Regulators, PloS Pathogens 2 (11): 1028-1030


Progress 01/01/05 to 12/31/05

Outputs
The overall goal of the project is to identify novel cis-elements and transcription factors controlling the plant defense transcriptome as well as their use to design new strategies to improve disease resistance in crops. We made important progress regarding both major objectives defined in the proposal. Objective 1.) a.): Using microarray data we defined two groups of Arabidopsis genes co-expressed in response to infections with Peronospora, EURP (early up-regulated in response to Peronospora recognition) and LURP (late up-regulated in response to Peronospora recognition). We identified several so far unknown promoter motifs significantly conserved in each gene set which may act as cis-elements responsible for their coordinated expression. In electrophoretic mobility shift assays five distinct conserved motifs exhibited strong Peronospora-inducible binding to proteins from Arabidopsis nuclei, suggesting interactions with defense-associated transcription factors. Objective 1.) b.): In addition we performed functional analyses of the LURP1, LURP2 and PAD4 promoters in transgenic Arabidopsis lines. We had finished cloning of 11 binary T-DNA vectors containing fusions of promoter deletions to GUS. Since our last report we have generated multiple independent transgenic lines with single insertions for each of these constructs. These lines were tested for GUS expression in response to Peronospora. None of the tested PAD4 promoter stretches conferred GUS expression after any of the tested treatments. Promoter stretches from LURP1 and LURP2 mediated strong GUS expression in response to Peronospora, but not or only weakly after control treatments. Deletion analysis limited important Peronospora-responsive cis-elements to a 114 bp stretch of the LURP1 promoter and a 252 bp stretch of the LURP2 promoter. Interestingly, the latter one does not contain any known plant cis-elements. Combined with results from Objective 1.) a.) a detailed dissection of these promoter stretches by reporter gene assays with additional promoter constructs will lead to the definition of novel pathogen-responsive cis-elements. Objective 2): Originally we proposed using the yeast one-hybrid system to clone transcription factors binding to promoter elements identified by methods described under Objective 1. We finished the construction of two separate cDNA/activation domain expression libraries representing transcripts from untreated and Peronospora-infected Arabidopsis seedlings. The titer of each primary library is 1xE6. We initiated screens of both libraries for factors binding to conserved motifs described above. We have also initiated DNA-affinity chromatography combined with Mass-Spectrometry to identify protein complexes binding to conserved promoter motifs as a second approach. This resulted in the identification of known or putative transcription factors specific for each tested conserved motif. We expect to identify some of these candidates also by our yeast one hybrid screens. Such transcription factors are likely to play important roles in the plant immune system and will be examined further using T-DNA mutants, overexpression or gene silencing.

Impacts
We examine molecular mechanisms mediating defense gene regulation in plants. Findings from this study will be used to design new strategies to improve disease resistance in crops. Besides leading to increased crop production our strategy is also likely to lead to a reduction of pesticide use. The latter effect may substantially impact the quality of the natural environment and conservation of natural resources.

Publications

  • - McDowell, J.M., Williams, S.G., Funderburg, N.T., Eulgem, T. and Dangl, J.L. (2005) Genetic Analysis of Developmentally Regulated Resistance to Downy Mildew (Hyaloperonospora parasitica) in Arabidopsis thaliana.; Molecular Plant Microbe Interactions, 18, (11), 1226 to 1234
  • - Eulgem, T. (2005) Regulation of the Arabidopsis Defense Transcriptome. Trends in Plant Sciences , 10 (2), 71 to 78


Progress 01/01/04 to 12/31/04

Outputs
I started as a new Assistant Professor at UCR in September 2003. Since then we have fully equipped our lab, recruited one post-Doc as well as one graduate student, set up most methods required for the proposed project (e.g. electrophoretic mobility shift assays (EMSAs), molecular cloning, transformation of Arabidopsis) and performed some key experiments. Besides the Post-Doc and the graduate student, a total of seven undergraduate students were involved in parts of the project. A visiting graduate student will join the project from February to July 2005. A second post-doc will start on a related project in March 2005. We made very important experimental progress. Using our own microarray data (partly published in Eulgem et al., 2004, Plant Physiol. 135:1129-44; Chen et al., 2002, Plant Cell, 14: 559-574; Teige et al. 2004, Molecular Cell, 15:141-52) as well as data from other labs (e.g. Tao et al., 2004, Plant Cell, 2003. 15: 317-30) we defined several sets of co-regulated defense genes. The algorithms AlignACE and Meme allowed us to identify multiple conserved promoter motifs in each gene set. Most importantly, by EMSA we identified nuclear DNA-binding activities that specifically interact with some of these conserved promoter motifs. We are currently focusing to reveal the identity of two of these DNA-binding activities that caused very intense band shifts in EMSAs. One of them interacts with a W box-like promoter motif and is induced after Peronospora recognition or treatment with the defense hormone salicylic acid. The second one constitutively interacts with a novel type of promoter motif. Binding to this novel motif is abolished after Peronospora recognition. These results demonstrate the feasibility of our approach using conserved promoter motifs to identify regulatory elements of the plant immune system. Yeast one hybrid screens to clone cDNAs corresponding to these DNA binding activities are being initiated. We are also in the process of purifying the respective proteins by DNA-Affinity Chromatography. Purified protein fractions will soon be analyzed by Mass-Spectrometry using the CEPCEB (Center for Plant Cell Biology) Proteomics facility. If strong cooperative interactions of components within a protein complex are essential for the observed DNA binding activities, cloning their isolated cDNAs by yeast one hybrid screening may not be successful. In this case, DNA-Affinity Chromatography combined with Mass-Spectrometry should allow identifying components of such a complex. EMSAs to identify nuclear proteins interacting with additional conserved promoter motifs are in progress. Furthermore, we finished the construction of a set of transgenic Arabidopsis lines for promoter analyses and will soon start a first round of reporter gene assays to examine promoters of three genes representing distinct Peronospora-induced expression patterns (CaBP22, PAD4 and At2g14500). The CaBP22 promoter, proved in EMSAs to differentially interact with a Peronospora-induced sequence specific DNA binding activity. By reporter gene assays we will perform a detailed functional dissection of this promoter and the other two promoters.

Impacts
We examine molecular mechanisms mediating defense gene regulation in plants. Findings from this study will be used to design new strategies to improve disease resistance in crops. Besides leading to increased crop production our strategy is also likely to lead to a reduction of pesticide use. The latter effect may substantially impact the quality of the natural environment and conservation of natural resources

Publications

  • Eulgem T, Weigman VJ, Chang HS, McDowell JM, Holub EB, Glazebrook J, Zhu T, Dangl JL. 2004. Gene expression signatures from three genetically separable resistance gene signaling pathways for downy mildew resistance. Plant Physiol. 135(2):1129-44.
  • Teige M, Scheikl E, Eulgem T, Doczi R, Ichimura K, et al. 2004. The MKK2 Pathway Mediates Cold- and Salt Stress Signaling in Arabidopsis. Molecular Cell 15(1):141-52.
  • Eulgem, T, Regulation of the plant defense transcriptome; 2005 Trends Plant Sci., in press.